Guanidino phenylalanin compounds used as urokinase inhibitors

ABSTRACT

The invention relates to the use of derivatives of 3-guanidinophenylalanine as urokinase inhibitors for treating malignant tumors and metastasis.

The invention relates to the use of derivatives of3-guanidinophenylalanine as urokinase inhibitors, in particular fortreating malignant tumors and metastases or as agents for targetinglymph cells, and for treating diseases of the lymphatic tissue, inparticular lymphomas.

The ability of solid tumors to spread into, and metastasize in,surrounding tissue correlates with the breakdown or remodeling of theextracellular matrix (tumor stroma) in the environment of the tumor cellor with its ability to penetrate the basement membrane. Although the(patho)biochemical circumstances have not yet been finally clarified,the plasminogen activator urokinase (uPA) and the urokinase receptor(uPAR) are considered to be of central importance. uPA mediates theproteolytic cleavage of plasminogen to give plasmin. Plasmin is in turna protease which has a broad spectrum of activity and is able todirectly break down the components of the extracellular matrix such asfibrin, fibronectin, laminin and the protein backbone of theproteoglycans. In addition, plasmin is able to activate “latent”metalloproteases and the inactive proenzyme of uPA, i.e. prb-uPA.

Tumor cells and nonmalignant cells of the tumor stroma synthesize andsecrete the enzymically inactive proenzyme pro-uPA. Proteases, such asplasmin or the cathepsins B and L, cleave pro-uPA, by means of limitedproteolysis, to give the active serine protease HMW-uPA (HMW=highmolecular weight). pro-uPA and the active protease HMW-uPA bind to thecell surface receptor uPAR (CD87). Plasmin(ogen) also binds to specificreceptors on the plasma membrane of the tumor cell, thereby focussingand amplifying plasminogen activation in the direct environment of thetumor cell. Invasive cells are consequently provided with thepossibility of breaking down the extracellular matrix withoutwithdrawing, by proteolysis, from the substrate which is required fordirectional movement.

A variety of cell-biological studies have shown that the cell-associatedplasminogen activator system is of special importance in thecascade-like reaction pathways of tumor-associated proteolysis systems(Wilhelm et al. (1994) The Urokinase/Urokinase receptor system: A newtarget for cancer therapy? In: Schmitt M., Graeff H., Kindermann G.(ed.): Prospects in Diagnosis and Treatment of Cancer. InternationalCongress Series, Excerpta Medica 1050, Amsterdam, Elsevier 1994, pp145-156). It has been observed that the ability of cultures of humancolon carcinoma cells to migrate through an extracellular matrix dependson the degree to which the uPA receptors are saturated with active uPA(Hollas et al., Cancer Res. 51 (1991), 3690-369.5). It has also beenobserved, in a cell culture model, that the invasive potential of cellsis reduced if the proteolytic activity of uPA is inhibited by PAI-1(Cajot et al., Proc. Natl. Acad. Sci. USA 87 (1990), 6939-6943) or PAI-2(Baker et al., Cancer Res. 50 (1990), 467.6-4684). A comparable effecthas been achieved by inhibiting the binding of uPA to the cell surfaceby blocking the receptor with proteolytically inactive uPA variants(Cohen et al., Blood 78 (1991), 479-487; Kobayashi et al.,. Br. J.Cancer 67 (1993), 537-544). Transfecting epidermoid carcinoma cells witha plasmid which was expressing an antisense transcript directed againsta part of uPAR also resulted, by suppression of uPAR synthesis, in adecrease in the invasiveness of these cells (Kook, EMBO J. 13 (1994),3983-3991). Antibodies directed against uPA and PAI-1 reduced theinvasive potential of lung cancer cells in vitro (Liu et al., Int. J.Cancer 60 (1995), 501-506). It has also been possible to confirm theinfluence of the plasminogen activatory system on the metastasis processin animal tumor models. Thus, the formation of lung metastases by humancarcinoma cells in chick embryos was almost completely prevented byadding antibodies directed against uPA (Ossowski and Reich, Cell 35(1983), 611-619). Metastasizing human carcinoma cells have beentransfected with an expression plasmid which encoded a proteolytically.Inactive, but uPAR-binding, uPA mutant. It was found in a mouse modelthat, following injection, the carcinoma cells which were synthesizinginactive uPA formed a significantly lower number of metastases than didthe untransfected cells (Crowley et al., Proc. Natl. Acad. Sci. USA 90(1993) 5021-5025). In addition, inhibition of the intraperitonealdissemination of human ovarian carcinoma cells was observed in nude micefollowing the administration of uPA antisense oligonucleotides (Wilhelmet al., Clin. Exp. Metast. 13 (1995), 296-302).

In recent years, the clinical relevance of factors of the plasminogenactivator system (uPA, uPAR, PAI-1 and PAI-2) for the prognosis ofpatients having solid malignant tumors has been investigatedintensively. In this connection, it has been found that the content ofuPA antigen in a variety of tumors (e.g. breast, ovary, stomach, lung,kidney, etc.) is a strong prognosis factor both for relapse-freesurvival and for mortality (see, for example, Schmitt et al., J. Obstet.Gynaecol. 21 (1995), 151-165; Jaenicke et al., Breast Cancer Res. Treat.24 (1993), 195-208; Kuhn et al., Gynecol. Oncol. 55 (1994), 401-409;Nekarda et al., Lancet 343 (1994), 117; Pedersen et al., Cancer Res. 54(1994), 4671-4675). Elevated concentrations of uPAR in lung (Pedersen etal., see above) and breast cancer tissue (Duggan et al., Int. J. Cancer61 (1995), 597-600; Ronne et al., Breast Cancer Res. Treat. 33 (1995),199-207), as well as in stomach cancer, both in the tumor tissue itself(Heiss et al., J. Clin. Oncol. 13 (1995), 2084-2093) and in the tumorcells which have disseminated into the bone marrow (Heiss et al., NatureMedicine 1 (1995), 1035-1039), likewise correlate with an unfavorableprognosis.

The use of synthetic uPA inhibitors offers the possibility ofsuppressing the invasion and dissemination of tumor cells. However, thedevelopment of specific uPA inhibitors is a difficult matter since thetissue-type plasminogen activator (tPA) has an identical specificity forcleaving the Arg560/Val561 peptide bond of plasminogen. In most cases,therefore, low molecular weight uPA inhibitors also inhibit tPA and, asa consequence, also inhibit tPA-mediated fibrinolysis. In addition, caremust be taken to ensure that synthetic uPA inhibitors do not exhibit anystrong inhibition of plasmin.

Despite these limitations, some inhibitors are known which possess acertain degree of specificity toward uPA but which have low inhibitorycapacity, such as benzamidine and β-naphthamidine derivatives, with themost active compound inhibiting uPA with a K_(i)=2.2 μmol/l(Stürzebecher and Markwardt, Pharmazie 33 (1978), 559), or amiloride,with a K_(i)=7 μmol/l (Vassalli and Belin, FEBS. Lett. 214 (1987),187-191).

DE-A-30 35 086 discloses cyclohexanecarboxylic acid derivatives whichhave inhibitory effects on proteases such as trypsin, chymotrypsin,thrombin and uPA. However, the investigated compounds only exhibit quitelow, and furthermore nonspecific, inhibition of uPA. EP-A-0 183 271discloses lysine derivatives and their use as protease inhibitors. Thisdocument also describes a benzamidinolysine derivative (compound 108)which inhibits uPA in vitro but also has a comparable effect on otherproteases such as trypsin or plasma kallikrein. WO 95/17885 discloseslow molecular weight polypeptides as being uPA inhibitors.

4-Substituted benzothiophene-2-carboxamidines, having a K_(i)=0.16μmmol/l in the case of benzothiophene-623, constitute another class ofknown uPA inhibitors (Towle et al., Cancer Res. 53 (1993), 2553-2559).These inhibitors have a significantly higher affinity for uPA than fortPA and plasmin. uPAR-bound uPA is also inhibited with higherefficiency. However, a disadvantage of these substances is that it is acomplicated matter to synthesize them chemically and that fewpossibilities for modifying the structure are available or have thus farbeen demonstrated.

Nα-Arylsulfonyl- and Nα-Arylsulfonylaminoacyl derivatives of3-amidinophenylalanine are known to be selective inhibitors of thrombin(Markwardt et al., Thromb. Res. 17 (1980), 425-431) and, respectively,coagulation factor Xa (Stürzebecher et al., Thromb. Res. 54 (1989),245-252). WO 92/08709 and WO 96/05189 also disclose the use ofamidinophenylalanine derivatives as inhibitors of blood coagulation, inparticular as inhibitors of thrombin. WO 94/18185 discloses amidino andguanidino derivatives of phenylalanine and their use as inhibitors ofblood coagulation, in particular as substances which have anantithrombotic effect.

Piperidides and piperazides of 3-amidinophenylalanine, among whichindicative structures for inhibiting fibrinolytic enzymes were alsofound, have been investigated intensively (Stürzebecher et al., J.Enzyme Inhibition 9, 87-99, 1995; Stürzebecher et al., J. Med. Chem. 40,3091-3099, 1997). While Stürzebecher et al. (1995) only describe theinhibition of thrombin, factor Xa, plasmin and trypsin, Stürzebecher etal. (1997) also contain information with regard to the inhibition ofuPA. In the case of Nα-2-naphthylsulfonyl-,Nα-2-(2,2,5,7,8-pentamethylchroman-6-yl)sulfonyl- andNα-2-camphor-10-ylsulfonyl-substituted3-amidinophenylalaninepiperazides, a K_(i) value of from 28 to 140μmol/l was found for uPA, with this K_(i) value being about three ordersof size higher than the inhibition constant for thrombin.

It has also been found that 3-amidinophenylalanine derivatives which aresubstituted by a phenyl radical at position 2 are selective inhibitorsof uPA which are active in vivo (PCT/EP99/05145). In addition, it hasbeen found that these substances exhibit a high degree of selectivityfor lymphatic tissue and are therefore suitable for the use as agentsfor targeting lymph cells, for example for treating malignant diseasesof the lymphatic tissue, such as lymphomas.

However, there is still a need to develop additional inhibitors whichhave a high degree of selectivity for uPA in order to further clarifythe role which uPA and uPAR play in various diseases, especially inconnection with tumor dissemination and metastasis.

Surprisingly, it has now been found that replacing the amidino functionwith the guanidino function in the phenylalanine derivatives does nothave a detrimental influence on the inhibitory effect in regard to uPAand that, furthermore, such inhibitors exhibit a high degree ofselectivity for uPA.

The present invention relates to novel 3-guanidino-phenylalanine-derivedurokinase inhibitors of the formula I,

which are present as racemates and as L- or D-configured compounds andin which

-   R1 (a) is OH or OR⁴, where R⁴ is an optionally, e.g. by hydroxyl,    carboxyl, sulfonyl, nitro, cyano, oxo and/or halogen, substituted,    branched or unbranched C₁-C₈-alkyl, C₃-C₈-cycloalkyl or aralkyl,    e.g. benzyl or phenylethyl,    -   (b) is a group of the formula    -    in which R⁵ and R⁶ are arbitrary radicals which are compatible        with the overall structure, where, in particular,        -   (i) R⁵ and R⁶ are H,        -   (ii) R⁵ is H and R⁶ is an optionally, e.g. by hydroxyl,            carboxyl, sulfonyl, nitro, cyano, oxo and/or halogen,            substituted, branched or unbranched C₁-C₈-alkyl, aralkyl,            e.g. benzyl or phenylethyl, or C₅-C₈-cycloalkyl,        -   (iii) R⁵ and R⁶ are in each case, independently, an            optionally, e.g. by hydroxyl and/or halogen, substituted,            unbranched or branched C₁-C₄-alkyl, or        -   (iv) R⁵ is H and R⁶ is —NH₂ or an amino group which is, in            particular, substituted by aryl or heteroaryl,        -   (v) R⁵ is H or an optionally, e.g. by hydroxyl and/or            halogen, substituted, unbranched or branched C₁-C₄-alkyl and            R⁶ is the radical of an amino acid, e.g. of an α-, β- or            ω-aminocarboxyl c or aminosulfonic acid, or the radical of a            peptide e.g. having a length of up to 50 amino acids or of a            polypeptide e.g. having a length of more than 50 amino acids            up to 1000 amino acids,    -   (c) is a group of the formula        -   in which m denotes the number 1 or 2 and in which one or            more of the methylene groups is/are optionally, e.g. by a            hydroxyl, carboxyl, C₁-C₄-alkyl or aralkyl radical, e.g.            benzyl or phenylethyl, substituted, where the group (c) is            racemic or D- or L-configured, and R⁷ has the meaning of R¹            in subsections (a), (b) and (f),    -   (d) is a group of the formula        -   in which p=r=1, p=1 and r=2 or p=2 and r=1 and in which one            or more of the methylene groups is/are optionally, e.g. by a            hydroxyl, carboxyl, C₁-C₄-alkyl or aralkyl radical, e.g.            benzyl or phenylethyl, substituted, and R⁷ has the meaning            of R¹ in subsections (a), (b) and (f),    -   (e) is a piperidyl group which is optionally substituted, e.g.        by a C₁-C₄-alkyl, c₁-C₃-alkoxy or hydroxyl radical, in one of        the positions 2, 3 and 4, where an additional aromatic or        cycloaliphatic ring, preferably phenyl or cyclohexyl, is        optionally fused, in the 2,3 or 3,4 position, based on the        heteroatom, to the heterocycloaliphatic rings of the formulae        (c), (d) and (e),    -   (f) is a group of the formula        -   in which R⁸        -   (i) is an optionally, e.g. by C₁-C₆-alkyl, C₁-C₃-alkoxy,            hydroxyl, carboxyl, sulfonyl, nitro, cyano, oxo and/or            halogen, substituted C₁-C₆-alkyl or aryl radical, such as            phenyl, p-halophenyl or naphthyl,        -   (ii) is a saturated or unsaturated, branched or unbranched            C₁-C₆-alkoxy radical,        -   (iii) is an oxycarbonyl radical —COOR′, in which R′ is H or            a group such as C₁-C₆-alkyl, aryl or aralkyl which is            optionally substituted by C₁-C₃-alkoxy, hydroxyl, carboxyl,            sulfonyl, nitro, cyano, oxo and/or halogen, e.g. an            ethoxycarbonyl, phenoxycarbonyl or benzyloxycarbonyl            radical, or        -   (iv) is an aminocarbonyl radical —CONR′R″, in which R′ and            R″ are in each case, independently, H or a group such as            C₁C₄-alkyl, aryl or aralkyl which is optionally substituted            by C₁-C₃-alkoxy, hydroxyl, carboxyl, sulfonyl, nitro, cyano,            oxo and/or halogen, e.g. ethylaminocarbonyl,    -   (g) is an acyl radical of the formula —COX, where X is        -   (i) H, an optionally, e.g. by hydroxyl, carboxyl, sulfonyl,            nitro, cyano, oxo and/or halogen, substituted, unbranched or            branched alkyl radical, preferably a C₁-C₆-alkyl radical, in            particular methyl,        -   (ii) an optionally, e.g. by C₁-C₆-alkyl, C₁-C₃-alkoxy,            hydroxyl, carboxyl, sulfonyl, nitro, cyano, oxo and/or            halogen, substituted aryl or heteroaryl radical such as            phenyl, p-halophenyl or thienyl, or        -   (iii) an optionally, e.g. by hydroxyl, carboxyl, sulfonyl,            nitro, cyano, oxo and/or halogen, substituted cycloalkyl            radical, preferably a C₃-C₁₀-cycloalkyl radical,    -   (h) is an aralkyl radical, e.g. benzyl or phenylethyl, in which        the aromatic radical is optionally substituted, e.g. by a        halogen atom or by a C₁-C₆-alkyl, C₁-C₃-alkoxy, hydroxyl, cyano,        carboxyl, sulfonyl or nitro group,    -   (i) is a carboxamide radical of the formula —CONR′R″, a        thiocarboxamide radical —CSNR′R″ or an acetamide radical        —CH₂—CONR′R″, where        -   (i) R′ and R″ are H,        -   (ii) R′ and R″ are in each case, independently, C₁-C₄-alkyl,        -   (iii) R′ is H and R″ is C₁-C₄-alkyl,        -   (iv) R′ is H and R″ is aryl, e.g. phenyl, or        -   (v) R′ and R″ form, together with the nitrogen atom, a            heterocycloaliphatic ring having 0.5-7 ring members which            can carry an additional heteroatom, e.g. N, O and/or S,    -   (j) is an SO₂—Y radical in which Y        -   (i) is an optionally, e.g. by hydroxyl, carboxyl, sulfonyl,            nitro, cyano, oxo and/or halogen, substituted C₁-C₈-alkyl,            preferably methyl, trifluoromethyl or trichloromethyl,        -   (ii) is an optionally, e.g. by C₁-C₆-alkyl, C₁-C₃-alkoxy,            hydroxyl, carboxyl, sulfonyl, nitro, cyano, oxo and/or            halogen, substituted aryl or heteroaryl, such as phenyl,            4-methylphenyl, 2,4,6-trimethylphenyl,            2,4,6-triisopropyl-phenyl, 4-methoxy-2,3,6-trimethylphenyl,            2,2-dimethyl-6-methoxychromanyl,            2,2,5,7,8-pentamethylchromanyl, anthra-quinonyl, naphthyl or            quinolyl, or O-aryl, preferably O-phenyl, or O-heteroaryl,            or        -   (iii) is —NR′R″, where R′ and R″ are in each case,            independently, H or C₁-C₃-alkyl,    -   (k) is a cycloaliphatic ring having 5 to 8 C atoms which is        optionally substituted, e.g. by a C₁-C₆-alkyl, C₁-C₃-alkoxy,        halogen, hydroxyl and/or oxo group,    -   (l) is an optionally, e.g. by C₁-C₆-alkyl, C₁-C₃-alkoxy,        hydroxyl, carboxyl, sulfonyl, nitro, cyano, oxo and/or halogen,        substituted heteroaryl radical, such as pyridyl or pyrimidyl, or        heterocycloaliphatic radical, for example N-methylpiperidyl,    -   (m) is a functionalized alkyl radical of the formula        —(CH₂)_(n)-X, where the alkyl chain is unbranched or branched,        n=1 to 8 and the functional radical X        -   (i) is a hydroxyl group whose H atom is optionally            substituted by a C₁-C₄-alkyl group, aralkyl group, e.g.            benzyl or phenylethyl, aryl group, e.g. phenyl,            C₁-C₄-hydroxyalkyl group or acyl group. CO-alkyl, (C₁-C₆),        -   (ii) is a halogen atom,        -   (iii) is a tertiary amino group of the formula —N(Alk)₂,            where the alkyl groups have 1 to 3 C atoms and also            preferably the same meaning, and the nitrogen atom            optionally belongs to a heterocyclo-aliphatic ring which has            5-7 ring members and can carry an additional heteroatom,            e.g. N, O and/or S,-   R² is an optionally, e.g. by C₁-C₆-alkyl, C₁-C₃-alkoxy, hydroxyl,    carboxyl, sulfonyl, nitro, cyano, oxo and/or halogen, substituted    phenyl radical, such as phenyl, 4-methylphenyl,    2,4,6-trimethylphenyl, 2,4,6-triisopropylphenyl or    4-methoxy-2,3,6-trimethylphenyl,-   R³ is H or branched or unbranched C₁-C₄-alkyl and n is 0 or 1,-   Z is N or CR⁹, where R⁹ is H or branched or unbranched C₁-C₄-alkyl.

The compounds can also be present as salts, preferably asphysiologically tolerated acid salts, e.g. as salts of mineral acids,particularly as hydrochlorides, or as salts of suitable organic acids.

Of the compounds which are defined in the general claims, those in whichR¹ corresponds to a group of the formulae (b), (d) and (f), R² is asingly, doubly or triply alkyl-substituted phenyl radical, in particulara 2,4,6-substituted phenyl radical, e.g. a 2,4,6-triisopropylphenylradical, and n=0, are of particular importance. Preference isfurthermore given to compounds in which Z is CH or N. R¹ is particularlypreferably a 4-ethoxycarbonyl piperazide radical or a4-ethylaminocarbanyl piperazide radical.

The present invention relates to the compounds of formula (I) as such,as active compounds in pharmaceutical compositions and for use forpreventing or treating diseases which are associated with urokinaseand/or urokinase receptor, in particular with their overexpression.

The compounds of the formula I can be prepared, and tested for theirin-vitro biological activity, in what is in principle a known manner, asdescribed, for example, in WO 92/08709 and WO 94/18185.

(L)-, (D)- or (D,L)-3-nitrophenylalanine is reacted with an appropriatesulfonyl chloride or a sulfonylated amino acid or its halide in thepresence of a base to give a compound of the formula I which possesses anitro function and in which. R¹═OH and R² and R³ and n correspond to themeanings defined in the general claims. The resulting compounds areconverted, by means of esterification with an appropriate alcohol underacid-catalytic conditions, into compounds of the formula I where R¹=(a).Using a method which is customary in peptide chemistry, e.g. a DCCmethod in the presence of HOBt, the compounds of the formula Ipossessing a corresponding R¹ can be prepared by reacting the carboxylicacids of the formula I (R¹═—OH) with a nucleophile of the structures(b), (e) and (f). For synthesizing compounds in which R¹=(c) and (d),the carboxylic acids of the formula I in which R¹═OH are initiallyreacted with cycloaliphatic amino acid esters of the structures (c) and(d), where R⁷ is preferably —OCH₃ and, respectively, —OC₂H₅, and theresulting carboxylic esters are hydrolyzed under mild acidic or alkalineconditions to give the corresponding carboxylic acids which cansubsequently be esterified in already described manner or reacted withnucleophiles of the structure (b), (e) or (f), with compounds of theformula I in which R¹=(c) and (d) and R⁷=(a), (b), (e) and (f) beingobtained.

Target compounds of the formula I containing a guanidine structure canbe obtained from the nitro compounds in a known manner, with thecorresponding amines as a rule initially being obtained by reduction ofthe nitro group, and with the amines then being converted, by reactionwith a suitable guanidinylating reagent, such as guanylpyrazole, intothe guanidino compounds.

Urokinase inhibitors according to the invention can be used, whereappropriate together with at least one suitable pharmaceutical auxiliaryor carrier substance, for producing drugs, e.g. drugs which can beadministered orally, subcutaneously or intravenously, for controllingtumors or in diagnosis. It is likewise possible to administer them incombination with other active compounds, e.g. other urokinase inhibitorssuch as antibodies and/or peptides.

The drugs for controlling tumors in humans and animals can beadministered topically, orally, rectally or parenterally, e.g.subcutaneously or intravenously, in the form of tablets, sugar-coatedtablets, capsules, pellets, suppositories, solutions or transdermalsystems such as plasters.

The compounds of the formula (I) are particularly preferablyNα-(2,4,6-triisopropylphenylsulfonyl)-3-guanidino-(D,L)-phenylalanine-4-ethoxycarbonylpiperazide andNα-(2,4,6-triisopropylphenylsulfonyl)-3-guanidino-(D,L)-phenylalanine-4-ethylaminopiperazideor the L-enantiomers thereof or pharmaceutically tolerated salts ofthese compounds, e.g. the hydrochlorides. These substances have goodsolubility behavior.

The compounds according to the invention are able to inhibit the growthand/or dissemination of malignant tumors, e.g. tumor dissemination inassociation with pancreatic carcinoma, tumor growth of the mammarycarcinoma and the metastasis of tumors, in a highly efficient manner. Inthis connection, the uPA inhibitors can, where appropriate, be employedtogether with other antitumor agents or with other types of treatment,e.g. irradiation or surgical interventions. In addition, the inhibitorsaccording to the invention are also active in the case of other uPA-and/or uPAR-associated diseases (e.g. for example in preventing blisterformation in connection with the skin disease pemphigus vulgaris).

uPA inhibitors according to the invention are preferably characterizedby the fact that they possess a K_(i) value for uPA which is at least 2times, preferably at least 5 times and particularly preferably at least10 times lower than that for plasmin, thrombin and/or tPA. It istherefore remarkable that the compounds according to the invention onlyhave a slight influence on blood coagulation and consequently exhibit asurprising degree of selectivity.

The substances according to the invention of the formula I can beemployed in the form of conjugates with physiologically activesubstances, e.g. together with radiolabels or with cytotoxic agents,e.g. chemotherapeutic agents, such as cisplatin, carboplatin,5-fluorouracil, doxorubicin, epirubicin or taxol, or peptides.Furthermore, the substances can also be incorporated into the membranesof carrier vesicles, e.g. liposomes, and consequently enable activesubstances, e.g. cytotoxic agents such as doxorubicin, which areenclosed in the carrier vesicles to be targeted.

The following example and figure are intended to clarify the invention.

FIG. 1 shows a scheme for synthesizing the substanceNα-(2,4,6-triisopropylphenylsulfonyl)-3-guanidino-(L)-phenylalanine-4-ethoxycarbonylpiperazine,which is preferred in accordance with the invention.

EXAMPLE 1 SynthesizingNα-2,4,6-triisopropylphenylsulfonyl-3-guanidino-(L)-phenylalanine-4-ethoxycarbonylpiperazinehydrochloride 1.1 N-Acetyl-(D/L)-(3-nitrophenylalanine)

A solution of 3-nitrobenzyl bromide (7.5 g; 35.7 mmol), diethylacetamidomalonate (7.54 g; 35.7 mmol) and potassium iodide (0.3 g, 1.79mmol) in abs. dioxane is stirred at 80° C. under argon while a solutionof sodium ethanolate (25 ml, 37.5 mmol) is added dropwise over a periodof 1.5 h. The reaction mixture is refluxed for 2 h and stirred overnightat RT. After 3M NaOH (24 ml) has been added at 80° C., the mixture isstirred at 95° C. for a further 4 h. The dioxane is distilled off andthe aqueous phase is extracted 3× with ethyl acetate (30 ml). Theaqueous phase is then acidified to pH 1 with 1M HCl and extracted 3×with ethyl acetate (30 ml). The product crystallizes out when the last 3pooled ethyl acetate phases are slowly evaporated. It is filtered offand dried in vacuo.

Yield: 6 g (69%), ESI-MS: m/z: 253.3 (M+H)⁺; calculated for C₁₁H₁₂N₂O₅:252.2

1.2 (L)-(3-Nitrophenylalanine)

A solution of N-acetyl-(D/L)-(3-nitrophenylalanine) (1.1) (5.75 g; 22.8mmol) in 350 ml of water is adjusted to pH 7.5 with 1M NaOH. After AmanoAcylase (0.25 g) has been added, the mixture is stirred slowly at 37-38°C. for 6 days. It is then acidified to pH 3 with 1M HCl and extracted 3×with ethyl acetate (in each case 100 ml); the organic phase isdiscarded. The pH of the aqueous solution is adjusted to 6.8 with 1MNaOH, after which the water is distilled off gradually and the product,which has precipitated, is filtered off and dried.

Yield: 1.7 g (35%), ESI-MS: m/z: 211.3 (M+H)⁺; calculated for C₉H₁₀N₂O₄:210.2

1.3 N-Triisopropylphenylsulfonyl-(L)-(3-nitrophenyl-alanine)

1M NaOH (8 ml) and a solution of triisopropylphenyl-sulfonyl chloride(2.42 g; 7.99 mmol) in dioxane (15 ml) are added dropwise in parallel,over a period of 1.5 h, to a solution of (L)-(3-nitrophenylalanine)(1.2) (1.7 g; 8.09 mmol) in a mixture of 1M NaOH (8 ml) and dioxane (15ml). The reaction mixture is then stirred at RT overnight. After thesolvent has been stripped off, the residue is taken up in ethyl acetate(50 ml) and this solution is washed 3× with a 5% solution of KHSO₄ (ineach case 25 ml) and 3× with water (in each case 20 ml). After thesolvent has been stripped off, the product is dried under high vacuum.

Yield: 3.52 g (92%), HPLC purity, approx. 80%; ESI-MS: m/z: 477.7(M+H)⁺; calculated for C₂₄H₃₂N₂O₆S₁: 476.6

1.4N-Triisopropylphenylsulfonyl-(L)-(3-nitrophenyl-alanine)-4-ethyloxycarbonylpiperazide

A solution of N-triisopropylphenylsulfonyl-(L)-(3-nitrophenylalanine)(1.3) (3.52 g; 7.39 mmol) 1-ethyloxycarbonylpiperazine (1.19 g; 7.47mmol) and HOBt (1.53 g; 11.3 mmol) in absolute DMF (15 ml) is cooleddown to 0° C. and a solution of DCC (1.7 g; 3.91 mmol) in DMF (10 ml) isadded dropwise. The reaction mixture is stirred at RT for 18 h. Afterthe solvent has been evaporated in vacuo down to approx. 7 ml, theresidue is diluted with ethyl acetate (50 ml) and this mixture is washed3× with a 5% solution of KHSO₄ and 3× with water. After the solvent hasbeen stripped off, the crude product is purified by flash chromatographythrough a silica gel column (gradient: petroleum ether:methyl tert-butylether (MTBE) 1:1 to MTBE:ethyl acetate 1:1).

Yield: 1.45 g (32%), ESI-MS: m/z: 617.9 (M+H)⁺; calculated forC₃₁H₄₄N₄O₇S₁: 616.8

1.5N-Triisopropylphenylsulfonyl-(L)-(3-aminophenyl-alanine)-4-ethyloxycarbonylpiperazide

A solution ofN-triisopropylphenylsulfonyl-(L)-(3-nitrophenylalanine)-4-ethyloxycarbonylpiperazide (1.4) (1.4 g; 2.27 mmol) in ethanol is hydrogenated, at RTfor 8 h, over 0.15 g of 10% Pd on active charcoal. After the catalysthas been filtered off, the solvent is stripped off in vacuo and theproduct is dried in +vacuo.

Yield: 1.35 g (98%), ESI-MS: m/z: 587.9 (M+H)⁺; calculated forC₃₁H₄₆N₄O₅S₁: 586.8

1.6N-Triisopropylphenylsulfonyl-(L)-(3-(N′N″bis-BOC-guanidino)phenylalanine)-4-ethyloxycarbonylpiperazide

A solution ofN-triisopropylphenylsulfonyl-(L)-(3-aminophenylalanine)-4-ethyloxycarbonylpiperazide (1.5) (0.5 g; 0.85 mmol) and N,N′-di-BOC-guanylpyrazole (0.27g; 0.85 mmol) in methylene chloride (10 ml) is stirred at RT for 6 dunder argon. After the solvent has been stripped off, the residue istaken up in ethyl acetate and this solution is extracted 3× with a 5%solution of KHSO₄ and 3× with a 5%-solution of NaHCO₃ (in each case 15ml). After the solvent has been stripped off, the crude product ispurified chromatographically through a silica gel column (gradient:petroleum ether (PE)/ethyl acetate (EA) 4:1 to PE/EA 7:3).

Yield: 0.6 g (85%), ESI-MS: m/z: 829.9 (M+H)⁺; calculated forC₄₂H₆₄N₆O₉S₁: 829.1

1.7N-Triisopropylphenylsulfonyl-(L)-(3-guanidino-phenylalanine)-4-ethyloxycarbonylpiperazide

N-Triisopropylphenylsulfonyl-(L)-(3-(N′N″bis-BOC-guanidino)phenylalanine)-4-ethyloxycarbonylpiperazide (1.6) (0.5 g; 0.603 mmol) is deprotected by stirring in 4MHCl in dioxane (8 ml). The solvent is stripped off and the residue istaken up in a little methanol; this solution is poured into MTBE (35ml), in connection with which the product precipitates out and issubsequently filtered off and dried in vacuo.

Yield: 0.18 g (49%), ESI-MS: m/z: 629.5 (M+H)⁺; calculated forC₃₂H₄₈N₆O₅S₁: 628.8; HPLC: t_(R)=6.8 min

The compounds were characterized mass spectrometrically using a Waters'ZQ 2000 ESI-MS mass spectrometer (Waters GmbH, Eschborn, Germany); apurity test was carried out by means of HPLC using an X-Terra C8 column,150×2.1 mm Ø (Waters GmbH, Eschborn, Germany), gradient: water/methanol50:50 to 5:95 in 15 min.

EXAMPLE 2 SynthesizingNα-2,4,6-triisopropylphenylsulfonyl-3-guanidino-(L)-phenylalanine-4-ethylaminocarbonylpiper-azinehydrochloride

The synthesis was effected in analogy with that described in Example 1apart from the fact that 1-ethylaminocarbonylpiperazine was used inplace of 1-ethyloxycarbonylpiperazine.

EXAMPLE 3

In-vitro inhibition of urokinase by selected compounds of the formula IConfiguration R¹ R² n K_(i)μmol/1 L

TIPP O 0.47

Abbreviations: TIPP-2,4,6-triisoprophylphenyl

Determining the Inhibitory Effect

In order to determine the activity of the inhibitor, 200 μl of Trisbuffer (0.05 mol/l, containing the inhibitor, 0.154 mol of NaCl/l, 5%ethanol, pH 8.0), 25 μl of substrate (Pefachrome UK orBz-βAla-Gly-Arg-pNA in H₂O; Pentapharm Ltd., Basle, Switzerland) and 50μl of sc-urokinase (Ribosepharm GmbH, Haan, Germany) were incubated at25° C. After 3 min, the reaction was terminated by adding 25 μl ofacetic acid (50%) and the absorption was determined at 405 nm using aMicroplate Reader (MR 5000, Dynatech, Denkendorf, Germany). The K_(i)values were determined in accordance with Dixon by means of linearregression using a computer program. The K_(i) values are the means ofat least 3 determinations; the standard deviation was less than 25%.

EXAMPLE 4

In-vitro inhibition of different serine proteases of the trypsin type byNα-(2,4,6-triisopropylphenyl-sulfonyl)-(L)-3-guanidinophenylalanine-4-ethoxycarbonylpiperazide (uPA inhibitor) K_(i) [μmol/l] Enzyme uPA Inh. urokinase 0.47plasmin 3.8 thrombin ≧12

The inhibitory effect for the enzymes employed was determined inaccordance with the principle described in Example 3.

It is evident from the values given above that the uPA inhibitoraccording to the invention surprisingly exhibits a smaller K_(i) valuethan plasmin by a factor of more than 5 and a K_(i) value for urokinasewhich is smaller than that for thrombin by a factor of more than 10.Consequently, the substances according to the invention are suitable foruse as selective urokinase inhibitors.

1. Use of compounds of formula I

which are present as racemates and also as D- or L-configured compoundsand in which R1 (a) is OH or OR⁴, where R⁴ is an optionally substituted,branched or unbranched C₁-C₈-alkyl, C₃-C₈-cycloalkyl or aralkyl, (b) isa group of the formula

 in which R⁵ and R⁶ are arbitrary radicals, where, in particular, (i) R⁵and R⁶ are H, (ii) R⁵ is H and R⁶ is an optionally substituted, branchedor unbranched C₁-C₈-alkyl, aralkyl or C₅-C₈-cycloalkyl, (iii) R⁵ and R⁶are in each case, independently, an optionally substituted, unbranchedor branched C₁-C₄-alkyl, or (iv) R⁵ is H and R⁶ is —NH₂ or an aminogroup which is, in particular, substituted by aryl or heteroaryl, (v) R⁵is H or an optionally substituted, unbranched or branched C₁-C₄-alkyl,or R⁶ is the radical of an amino acid, of a peptide or of a polypeptide,(c) is a group of the formula

 in which m denotes the number 1 or 2 and in which one or more of themethylene groups is/are optionally substituted, where the group (c) isracemic or D-configured or L-configured, and R⁷ has the meaning of R¹ insubsections (a), (b) and (f), (d) is a group of the formula

 in which p=r=1, p=1 and r 2 or p=2 and r=1 and in which one or more ofthe methylene groups is/are optionally substituted, and R⁷ has themeaning of R¹ in subsections (a), (b) and (f), (e) is a piperidyl groupwhich is optionally substituted in one of the positions 2, 3 and 4,where an additional aromatic or cycloaliphatic ring is optionally fused,in the 2, 3 or 3,4 position, based on the heteroatom, to theheterocycloaliphatic rings of the formulae (c), (d) and (e), (f) is agroup of the formula

 in which R⁸ (i) is an optionally substituted C₁-C₆-alkyl radical oraryl radical, (ii) is a saturated or unsaturated, branched or unbranchedC₁-C₆-alkoxy radical, (iii) is an optionally substituted oxycarbonylradical e.g. an ethoxy-carbonyl, phenoxycarbonyl or benzyl-oxycarbonylradical, or (iv) is an optionally substituted aminocarbonyl radical,e.g. an ethyl-aminocarbonyl radical, (g) is an acyl radical of theformula —COX, where X (i) is H or an optionally substituted, unbranchedor branched alkyl radical, (ii) is an optionally substituted aryl orheteroaryl radical, or (iii) is an optionally substituted cycloalkylradical, (h) is an aralkyl radical in which the aromatic radical isoptionally substituted, (i) is a carboxamide radical of the formula—CONR′R″, a thiocarboxamide radical —CSNR′R″ or an acetamide radical—CH₂—CONR′R″, where (i) R′ and R″ are H, (ii) R′ and R″ are in eachcase, independently, C₁-C₄-alkyl, (iii) R′ is H and R″ is C₁-C₄-alkyl,(iv) R′ is H and R″ is aryl, or (v) R′ and R″ form, together with thenitrogen atom, a heterocycloaliphatic ring which has 5-7 ring membersand which can carry a further heteroatom, (j) is a SO₂—Y radical inwhich Y (i) is an optionally substituted C₁-C₈-alkyl, (ii) is anoptionally substituted aryl or heteroaryl or O-aryl or O-heteroaryl, or(iii) is —NR′R″, where R′ and R″ are, in each case, independently, H orC₁-C₃-alkyl, (k) is a cycloaliphatic ring which has 5 to 8 C atoms andwhich is optionally substituted, (l) is an optionally substitutedheteroaryl radical or heterocycloaliphatic radical, (m) is afunctionalized alkyl radical of the formula —(CH₂)_(n)—X, where thealkyl chain is unbranched or branched, n=1 to 8 and the functionalradical X (i) is a hydroxyl group whose H atom is optionally substitutedby a C₁-C₄-alkyl group, aralkyl group, e.g. benzyl or phenylethyl, arylgroup, C₁-C₄-hydroxyalkyl group or acyl group CO-alkyl (C₁-C₆), (ii) isa halogen atom, (iii) is a tertiary amino group of the formula —N(Alk)₂,where the alkyl groups have 1 to 3 C atoms and the nitrogen atomoptionally belongs to a heterocycloaliphatic ring which has 5-7 ringmembers and which can carry an additional heteroatom S, R² is anoptionally substituted phenyl radical, R³ is H or branched or unbranchedC₁-C₄-alkyl and n is 0 or 1, Z is N or CR⁹, where R⁹ is H or branched orunbranched C₁-C₄-alkyl, or of salts of the compounds for producing anagent for diagnosing, treating and preventing urokinase-associated orurokinase receptor-associated diseases.
 2. The use as claimed in claim1, characterized in that R¹ is a group of the formulae (b), (d) and (f),R² is a 2,4,6-triisopropylphenyl radical and n=O.
 3. The use as claimedin claim 1, characterized in that the compound of the formula I isNa-(2,4,6-triisopropylphenylsulfonyl)-3-guanidino-(D,L)-phenylalanine-4-ethoxycarbonylpiperazide,Na-(2,4,6-triisopropylphenylsulfonyl)-3-guanidino-(D,L)-phenylalanine-4-ethylaminocarbonylpiperazide, or the L-enantiomer or a pharmaceutically tolerated salt ofone of the compounds.
 4. The use as claimed in claim 1, characterized inthat the compounds are present in the form of physiologically toleratedacid salts, in particular as hydrochlorides.
 5. The use as claimed inclaim 1 for controlling tumors.
 6. The use as claimed in claim 5 forcontrolling mammary carcinomas, pancreatic carcinomas and metastasisformation.
 7. The use as claimed in claim 1 for controlling pemphigusvulgaris.
 8. The use as claimed in claim 1, characterized in that thecompounds of formula I are employed as conjugates with otherpharmacologically active substances.
 9. The use as claimed in claim 1,characterized in that the compounds of formula I are employed incombination with other pharmacologically active substances.
 10. The useas claimed in claim 8, characterized in that the compounds are employedas conjugates with radiolabels and/or in combination with cytotoxicsubstances.
 11. The use as claimed in claim 1 for producing drugs whichcan be administered orally, topically, rectally or parenterally.
 12. Theuse as claimed in claim 1 in the form of tablets, sugar-coated tablets,capsules, pellets, suppositories, solutions or transdermal systems suchas plasters.
 13. A method for inhibiting urokinase in living beings, inparticular humans, by administering an effective quantity of at leastone urokinase inhibitor as claimed in claim
 1. 14. A compound of theformula I

in which R¹, R², R³ Z and n are defined as in claim
 1. 15.Na-(2,4,6-triisopropylphenylsulfonyl)-3-guanidino-(D,L)-phenylalanine-4-ethoxycarbonyl piperazide,Na-(2,4,6-triisopropylphenylsulfonyl)-3-guanidino-(D,L)-phenylalanine-4-ethylaminocarbonylpiperazide or the L-enantiomer thereof, or a pharmaceutically toleratedsalt of one of the compounds.
 16. A pharmaceutical composition,characterized in that it comprises, as active compound, one or morecompounds as claimed in claim 14, where appropriate together withpharmaceutically customary excipients, adjuvants and/or diluents.